PROJECT SUMMARY There are approximately 850,000 new cases of sepsis each year with mortality rates ranging from 240,000- 375,000. An impaired innate immune response can aggravate the septic condition by compromising the patient?s ability to combat an infection. However, the cells and mediators that enhance the innate immune response in sepsis are still unknown. Basophils account for less than 1% of peripheral blood leukocytes, which makes them the rarest known granulocytes. Basophils are evolutionarily conserved in many animal species, suggesting a beneficial rather than deleterious role of basophils. Nevertheless, it is unknown whether basophils play any role in the host?s defense against bacteria that can potentially prevent sepsis development. Our preliminary studies support such a role by showing that basophils are one of the very first cells to accumulate at the infection site at early stages of infection, and can improve survival and bacteria clearance in the polymicrobial model of sepsis induced by cecal ligation and puncture (CLP). We think that our findings in the murine system may be translatable to humans because we observed that trauma patients show increased numbers of basophils in circulation when a nosocomial infection was circumscribed to local tissues (early stages of infection) while basophil numbers decreased or remain unchanged when a patient developed a systemic infection (bacteremia) and was therefore at high risk of developing sepsis. Based on these studies, we hypothesize that basophils play a protective role in sepsis by enhancing the innate immune response against infection. Accordingly, we propose a research plan aimed at investigating the contribution of basophils to the innate immune response against bacteria. In Aim 1, we will identify mechanisms involved in basophil activation during an infection. We will use a genetic approach to investigate whether basophil stimulation through the TLR and MyD88 pathways is required to induce basophil activation and to confer protection during an infection; and we will examine whether the epithelial cell-derived cytokine, thymic stromal lymphopoietin (TSLP), can enhance the ability of basophils to respond to an infection. In Aim 2, we will define the mechanisms by which basophils confer protection against bacterial infections. Specifically, we will investigate interactions between basophils, the endothelium, and circulating leukocytes in a microvessel system and we will use mice with basophil-specific TNF deficiency to study these interactions during CLP. In Aim 3, we will establish the relevance of basophils in human infections and sepsis. Specifically, we will use mass cytometry (CyTOF) to assess basophil immune functions in samples collected from patients that develop nosocomial infections, mainly pneumonia, and we will establish whether these immune functions associate with clinical outcomes. We think that the studies proposed will expand our knowledge of sepsis physiopathology. Specifically, our studies will provide, for the first time, evidence for a critical role for basophils in the enhancement of the innate immune response against bacteria, an unexpected role for this rare cell population.